The brush border (BB) Na/H exchanger NHE3 is rapidly up and down regulated as part of digestion, with[unreadable] prolonged inhibition contributing to the pathophysiology of diarrhea. NHE3 exists in the BB in large,[unreadable] multiprotein complexes of varying size. In these complexes, NHE3 associates with 4 BB PDZ domain[unreadable] containing proteins, NHERF1, NHERF2, PDZK1 and IKEPP. The BB PDZ proteins are in somewhat different[unreadable] locations in Na absorptive cells, although each has a BB component. This project will test the hypotheses[unreadable] that acute NHE3 regulation in intestinal BB requires its presence in large multiprotein complexes scaffolded[unreadable] by these BB PDZ proteins; and that these complexes are dynamic and change in their location and makeup[unreadable] as part of NHE3 regulation. Proposed studies will knock down (shRNAi in Caco-2 cells) and out (gene[unreadable] targeting in mice) each of these BB PDZ proteins alone and in combinations. Physiologic and[unreadable] pathophysiologic regulation of NHE3 in mouse ileum and Caco-2 cells will be evaluated using the Ussing[unreadable] chamber/voltage clamp approach and two-photon microscopy with SNARF-4F to measure intracellular pH.[unreadable] Basal and acutely stimulated and inhibited NHE3 activity will be examined, with determination whether[unreadable] trafficking of NHE3 is affected; what size complexes in the BB NHE3 and the BB PDZ proteins are in, using[unreadable] sucrose density gradient centrifugation, under these conditions; and whether NHE3/NHERF proteins change[unreadable] their direct association as part of NHE3 regulation as assessed by FRET. Knockout/down models of the 4[unreadable] BB PDZ proteins (NHERF1, NHERF2 already on hand and PDZK1 is available) will be evaluated to[unreadable] understand their contribution to NHE3 regulation. NHE3 associates with the cytoskeleton directly by ezrin[unreadable] binding and indirectly by binding to ezrin via binding NHERF1, NHERF2 and PDZK1. Point mutations of[unreadable] NHE3 have been identified which separately eliminate this direct and indirect ezrin binding. These mutants[unreadable] will be expressed in Caco-2 cells and effects determined on NHE3 BB localization and on NHE3 basal and[unreadable] regulated activity, NHE3 complex formation and associating proteins, as well as on NHE3 mobility in the BB[unreadable] as assessed by FRAP. These studies will provide insight into how salt is absorbed normally in the intestine[unreadable] and becomes abnormal in diarrheal diseases.